Dynamic changes in intracellular pH (pHi) regulate a range of normal and pathological cell processes. Increased pHi promotes cell proliferation, differentiation, and migration, and decreased pHi induces apoptosis. Dysregulated pHi is thought to contribute to cancer progression, diabetes, and tissue damage after cardiac and cerebral ischemia. The long-term goals of this grant are to determine the regulation and function of pHi dynamics. The past four funding cycles focused on how H+ fluxes by the plasma membrane Na-H exchanger NHE1 are regulated and drive pHi dynamics. We also showed the functional significance of NHE1-dependent increases in pHi for cell proliferation, cell cycle progression, and cell migration. Despite the broad significance of pHi-dependent cell functions, we have limited understanding of how changes in pHi affect proteins and macromolecular assemblies. To address this limitation we recently began studying the structure and function of pH sensors, or proteins with activities or binding affinities that are sensitive to small physiological changes in pH. The current application applies what we collectively learned in the previous four funding cycles to determine the design principles and function of pH sensors regulating basic cell processes that are aberrant in cancer cells. Increased pHi is a hallmark of most cancers, regardless of the tissue origin or genetic background. This likely reflects a dependence on higher pHi for metabolic adaptation, increased proliferation, and metastasis. We will test the hypothesis that pH sensors controlling metabolism and cell cycle progression play critical roles in how NHE1 and pHi direct cell functions that are dysregulated in cancer. Our studies use an innovative comprehensive approach that bridges protein structure, protein biochemistry, and cell physiology. In Aim 1 we will determine how kinases controlling metabolism are regulated by NHE1. We will test predictions on how phosphoinositide 3-kinase is pH sensitive, based on our recent structural and biochemical findings that its p85 regulatory subunit is a pH sensor. We also will determine NHE1 regulation of phosphofructokinase 1 (PFK1), the first rate-limiting enzyme in glycolysis, which directly binds the C-terminal cytoplasmic domain of NHE1 and has extremely pH-sensitive activity. In Aim 2 we will determine how NHE1 promotes cell proliferation by focusing on our previous findings that H+ efflux by NHE1 times G2/M. We will test predictions on Wee1 as a putative pH sensor based on our new computational and biochemical data and on how cyclin B1 expression is attenuated in cells lacking NHE1 activity. In Aim 3 we will determine the role of NHE activity and dysregulated pHi in tumorigenesis using Drosophila models we generated. We will test predictions on how loss of Dnhe2 may be a synthetic lethal for transformed but not normal cells, and test rescue of phenotypes with mutant pH sensors generated in Aim 1 and 2. We also will ask whether over expression of Dnhe2 cooperates with oncogene activation or tumor suppressor deletion to induce metastatic cancer, and will use modifier screens to identify mediators of the Dnhe2 over expression phenotype.